Active cellular suicide or programmed cell death, also known as apoptosis, plays an important role in animal development, tissue homeostasis, immune response and a wide variety of pathological conditions including cancer, atherovascular diseases, diabetes, Alzheimer's disease, Parkinson's disease, stroke, severe infections, sepsis, renal failure, HIV/AIDS, autoimmune disorders etc. [Wyllie, A. H., Kerr, J. F. R., Currie, A. R. Cell death: the significance of apoptosis. Int. Rev. Cytol 68, 251-306 (1980); Fadeel, B., Orrenius, S., Zhivotovsky, B. Apoptosis in human disease: a new skin for the old ceremony. Biochem. Biophys. Res. Com. 266, 699-717 (1999)]. Apoptosis is characterized by programmed or systematic activation of a number of genes, especially those coding for caspases, which lead to cleavage of the chromatin/DNA into smaller fragments which are entrapped in apoptotic bodies that result from disintegration of the apoptotic cells. Under physiological conditions these apoptotic bodies and the chromatin/DNA contained within them are efficiently removed when ingested by macrophages.
Hundreds of billions of cells die in the body everyday and an equal number of cells are generated to replace them [Fliedner T. M., Graessle D, Paulsen C. & Reimers K. Structure and functions of bone marrow hemopoiesis: Mechanisms of response to ionizing radiation exposure. Cancer Biotherapy & Radio pharmaceuticals 17, 405-425 (2002)]. Unless these apoptotic cells are efficiently eliminated by phagocytosis, apoptotic chromatin/DNA can enter the blood stream from tissues and blood cells undergoing normal apoptotic turnover. Indeed, with the recent availability of a quantitative sandwich-enzyme-immunoassay which employs antibodies to both DNA and histones (Cell Death Detection ELISAPlus, Roche Biochemicals), fragments of chromatin in the form of mono- and oligonucleosomes have been shown to be present in sera of normal persons, and in higher quantities in patients with cancer, systemic autoimmune disorders, diabete's, cerebral stroke, myocardial infarction, inflammation, sepsis, critical illness, trauma, pulmonary embolism, inflammatory bowel disease, organ transplantation and pre-eclampsia. Leon S , Shapiro B, Sklaroff D M. et. al. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 1977 March; 37(3):646-50; Holdenrieder S, Stieber P, Bodenmuller H. et. al. Nucleosomes in serum of patients with benign and malignant diseases. Int J Cancer. 2001 Mar. 20; 95(2):114-20; Kremer A, Wilkowski R, Holdenrieder S. et. al. Nucleosomes in pancreatic cancer patients during radiochemotherapy. Tumour Biol. 2005 January-February; 26(1):44-9; Kremer A, Holdenrieder S, Stieber P. et. al. Nucleosomes in colorectal cancer patients during radiochemotherapy. Tumour Biol. 2006; 27(5):235-42; Butt A N, Shalchi Z, Hamaoui K. et. al. Circulating nucleic acids and diabetic complications. Ann NY Acad Sci. 2006 September; 1075:258-70; Zeerleder S, Zwart B, Wuillemin W A. et. al. Elevated nucleosome levels in systemic inflammation ‘and sepsis. Crit Care Med. 2003 July; 31(7):1947-51; Saukkonl:ln K, Lakkisto P, Pettila V. et. al. Cell-Free Plasma DNA as a Predictor of Outcome in Severe Sepsis and Septic Shock. Clin Chem. 2008 Apr. 17; Geiger S, Holdenrieder S, Stieber P. et. al. Nucleosomes in serum of patients with early cerebral stroke. Cerebrovasc Dis. 2006; 21 (1-2):32-7; Chang C P, Chia R H, Wu T L. et. al. Elevated cell-free serum DNA detected in patients with myocardial infarction. Clin Chim Acta. 2003 January; 327(1-2):95-1 01; Lam N Y, Rainer T H, Chan L Y. et.al. Time course of early and late changes in plasma DNA in trauma patients. Clin Chem. 2003 August; 49(8): 1286-91; Rainer T H, Lam N Y. Circulating nucleic acids and critical illness. Ann NY Acad Sci. 2006 September; 1075:271-7; Barada F A Jr, Suratt P M, Davis JS 4th. et. al. Free plasma DNA in patients with pulmonary embolism. South Med J. 1980. March; 73(3):345-6, 350; Galeazzi M, Morozzi G, Piccini M. et. al. Dosage and characterization of circulating DNA: present usage and possible applications in systemic autoimmune disorders. Autoimmun Rev. 2003 January; 2(1):50-5; Holdenrieder S, Eichhorn P, Beuers U. et. al. Nucleosomal DNA fragments in autoimmune diseases. Ann N Y Acad Sci. 2006 September; 1075:318-27; Benoit M, Fenollar F, Raoult D. et.al. Increased levels of circulating IL-16 and apoptosis markers are related to the activity of Whipple's disease. PLoS ONE. 2007 Jun. 6; 2(6):e494; Zhong X Y, Gebhardt S, Hillermann R. et. al. Circulatory nucleosome levels are significantly increased in early and late-onset preeclampsia. Prenat Diagn. 2005 August; 25(8):700-3.
It has been demonstrated that in patients with cancer, the elevated basal level of CCFs rises further following chemotherapy or radiotherapy within 24-72 hours [Holdenriedm, S. et al. Nucleosomes in serum of patients with benign and malignant diseases. Int J Cancer 95, 114-120 (2001)].
Blood component therapy/transfusion is a common therapeutic procedure. Since apoptotic chromatin fragments are known to circulate in blood of normal individuals, it is possible that during transfusion of blood or blood products such apoptotic chromatin fragments are transferred to the recipient leading to an increase in the burden of CCFs.
Apoptotic bodies can be ingested also by non-macrophage cells, such as fibroblasts, which are incapable of efficiently clearing them from the body. [Patnaik, R., Raff, M. C. & Scholes, J. Differences between the clearance of apoptotic cells by professional and non-professional phagocytes. Curr. Biol. 10, 857-860 (2000)]. When ingested by macrophages, the engulfed chromatin/DNA is known to be degraded and ultimately lost with the death of the scavenging cells. However, the fate of non-macrophage cells after they engulf the apoptotic chromatin fragments was unknown until recently.
It was recently shown that when CCFs purified from plasma/serum of normal subjects and patients suffering from different malignancies, both before and after chemo- or radiotherapy, are added to a variety of cells in culture, they freely enter the recipient cells without assistance and induce a DNA damage response (DDR) that is detectable within one hour. The DDR results in incorporation of exogenous chromatin into the host cell genomes. A DDR induced by chromatin fragments is observed in all cell types tested, including those of mesenchymal, epithelial, neuronal, endothelial, myocardial, hepatic and adipose tissue origin, as well as in isolated lymphocytes, suggesting that apoptotic chromatin may be an universal DNA damaging agent (I. Mittra, U. Samant, G. K. Modi, P. K. Mishra and G. S. Bhuvaneswar. A method for ex-vivo separation of apoptotic chromatin fragments from blood or plasma for prevention and treatment of diverse human diseases. US Patent Application No. FPAA819PCT dated Oct. 27, 2006).
When cells were exposed to purified CCFs, rapid onset of a sequence of morphological changes occurred in all cell types within a span of 7 days. The typical sequence included: cell cycle arrest→Increase in cell size→apoptosis→senescence of remaining cells. The PCCF treated cells showed numerous chromosomal aberrations as well as increase in size and number of centrosomes within 48 hours indicating the induction of chromosomal instability. By day 10, groups of rapidly proliferating, non-senescent cells with altered morphologies, some of which were apparently oncogenically transformed, arose and surrounded the senescent cells. When chromatin treated cells were injected into immuno-deficient mice, tumor development was observed in a proportion of the injected animals (I. Mittra, U. Samant, G. K. Modi, P. K. Mishra and G. S. Bhuvaneswar. A method for ex-vivo separation of apoptotic chromatin fragments from blood or plasma for prevention and treatment of diverse human diseases. US Patent Application No. FPAA819PCT dated Oct. 27, 2006).
When the effect of plasma was tested on cultured recipient cells and analysed by flow-cytometry using the apoptosis marker annexin V, induction of a much higher degree of apoptosis was seen in response to plasma obtained from patients suffering from diabetes, renal failure, sepsis and cancer, specially after chemo- or radiotherapy, compared to that induced by plasma from healthy subjects. When chromatin contained in plasma was removed by immuno-adsorption using anti-histone antibodies, the apoptosis inducing property of plasma was greatly attenuated (I. Mittra, U. Samant, G. K. Modi, P. K. Mishra and G. S. Bhuvaneswar. A method for ex-vivo separation of apoptotic chromatin fragments from blood or plasma for prevention and treatment of diverse human diseases. US Patent Application No. FPAA819PCT dated Oct. 27, 2006).
Progressive DNA damage leading to genomic instability, senescence and apoptosis of cells underlies human ageing [Kirkwood T. B. L. Understanding the odd science of aging. Cell 120, 437-447 (2005)]. Although free radicals generated within the body have been implicated as the DNA damaging agent related to ageing, this theory has not been satisfactorily substantiated [Lombard D.B. et al. DNA repair, genome stability, and aging Cell 120, 497-512 (2005)]. Increased DNA damage and apoptosis is also associated with a wide variety of age related degenerative diseases such as Alzheimer's disease, Parkinson's disease, Stroke, Atherovascular diseases, Diabetes etc. [Jellinger K. A. Cell death mechanisms in neurodegeneration. J Cell Mol Med 5, 1-17 (2001); Bennett M. R. Apoptosis in the cardiovascular system. Heart 87, 480-487 (2002); Otton R, Soriano F G, Verlengia R, Curi R. Diabetes induces apoptosis in lymphocytes. J Endocrinol 182, 145-56 (2004)].
Increased cellular apoptosis is also associated with inflammatory processes such as infections, sepsis, multi-organ system failure as well as autoimmune disorders [Hotchkiss H S et al. Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med. 27, 1230-1251(1999); Apoptosis and Autoimmunity from Mechanisms to Treatment, Edited by J. R. Kalden and M. Herrmann. Co. Wiley-Vch, Weinheim (2003);]. The above conditions are known to be associated with high circulating levels of apoptotic chromatin fragments in blood. [Zeerleder S et al. Elevated nucleosome levels in systemic inflammation and sepsis. Crit. Care Med. 31, 1947-1951 (2003) ; Williams, R. C., Malone, C. C., Meyers, C., Decker, P., Muller, S. Detection of nucleosome particles in serum and plasma from patients with systemic lupus erythematosus using monoclonal antibody 4H7. J Rheumatol 28, 81-94 (2001)]. It has been reported that renal failure is associated with an increased apoptotic turnover which may contribute to the high mortality in this condition. [D′Intini V et. al. Longitudinal study of apoptosis in chronic uremic patients. Semin Dial, 16,467-73 (2003); U.S. Renal Data System, USRDS 2005 Annual Data Report: Atlas of End-Stage Renal Disease in the United States, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Md., 2006]. HIV infection/AIDS is also associated with extremely high apoptotic turnover in CD4 positive cells and is causally related to the multiple pathological consequences/complications of this disease. [Badley A D, Pilon A A, Landay A, Lynch DH. Mechanisms of HIV-associated lymphocyte apoptosis. Blood,96:2951-64(2000) ]. Blood and blood products, that are routinely transfused for diverse medical indications, are known to be associated with an array of adverse consequences [Dellinger E P, Anaya D. A Infectious and immunologic consequences of blood transfusion. Critical Care 8, S18-S23 (2004)]. Transfusion of blood or blood products can increase the apoptotic chromatin burden in the recipient by i) delivering the existing apoptotic chromatin in the donor blood/blood products, ii) delivering apoptotic chromatin fragments that are derived from cells that undergo apoptosis during storage and processing. This chromatin overload may have deleterious effects on the recipient.
Based on the above findings, an ex-vivo system for removal of CCFs was proposed for the prevention/treatment of pathological conditions associated with increased DNA damage, genomic instability, senescence, apoptosis and oncogenic transformation. Such an ex-vivo system may be used for the prevention/retardation of spread of cancer, systemic autoimmune disorders, diabetes, Parkinson's disease, Alzheimer's disease, cerebral stroke, myocardial infarction, inflammation, sepsis, critical illness, trauma, renal failure, HIV/AIDS, etc. as well as ageing and other age-related disorders. (I. Mittra, U. Samant, G. K. Modi, P. K. Mishra and G. S. Bhuvaneswar. A method for ex-vivo separation of apoptotic chromatin fragments from blood or plasma for prevention and treatment of diverse human diseases. US Patent Application No. FPAA819PCT dated Oct. 27, 2006).
A method of treatment for systemic lupus erythematosus (SLE) is described by Diamond et al. (U.S. Pat No. 4,690,905). It uses monoclonal antibodies against anti-DNA antibodies and then using anti-idiotypic antibodies to remove the pathogenic anti-DNA antibodies from the patient's system. There are several drawbacks to this approach; it is dangerous to remove large quantities of blood from the patient. Removed blood has to be treated to remove the anti-DNA antibodies and then the treated blood is returned to the patient. It would be similar to hemodialysis, i.e., via an arterial passage. A risk of infection or the possible spreading of dangerous diseases such as HIV, hepatitis B, and hepatitis C is also there. Usage of nanoparticles avoids these problems.
High dose intravenous immune globulin (IVIG) infusions have also been used in treating certain autoimmune diseases. Previous studies have indicated that IVIG may contain anti-idiotype activity against anti-DNA antibodies, as well as many other autoantibodies (Jordan, S. C., 1989; Silvestris et al., 1994; Mouthon et al., 1996; Silvestris et al., 1996). The effects of IVIG infusions are apparently related to changes in the repertoire of autoantibodies expressed in the patient. This modulation of pathogenic Id antibodies is thought to depend on their specific interaction with the regulatory anti-idiotype molecules that occur naturally in healthy donors. Production of anti-idiotypic antibodies inhibiting the potentially harmful autoimmune repertoire may result from activation of the Id network committed to controlling the secretion of natural autoantibodies by CD5-positive B cells.
Treatment of SLE with IVIG has provided mixed results, including both resolution of lupus nephritis (Akashi et al., 1990), and in a few instances, exacerbation of proteinuria and kidney damage (Jordan et al., 1989). The cause of this increase is not clear but it is believed that there is increased glomerular deposition of immune-complexed, polyreactive, non-Id-specific IgG antibodies. Although there are several treatments for autoimmune disease such as SLE , all possess serious disadvantages.